| Editorial Summary |
(Editorial
Overview Text Replicated on Overall Disease page - Foot and
Mouth Disease) Killed (inactivated) vaccines against FMDV are produced by
growing virus in cell culture, inactivating the virus and combining it with an adjuvant, a
substance which enhances the immune response. Further processing may be carried out to
concentrate the antigen to reduce the volume required for vaccination of each animal, and
allow storage of antigen for prolonged periods without loss of efficacy. Testing during
vaccine production ensures safety and efficacy of vaccines produced under standards of
Good Manufacturing Practice.
The development and use of vaccines against foot-and-mouth disease virus
(FMDV) has been complicated by the presence of seven different immunologically
distinct virus types, antigenic variation within virus types, the continuous emergence of
new subtypes, and the relatively short time for which FMD vaccines provide effective
protection.
Considerable progress has been made in areas such as virus growth,
inactivation, purification and the development of effective adjuvants to boost the immune
response to the vaccine.
In recent years "vaccine banks" have been developed containing purified
concentrated vaccine which may be used to produce high potency emergency vaccines at short
notice.
The effectiveness of vaccination in practice depends not only on the standard of the
vaccine but also on its being stored, transported and administered correctly.
The effectiveness of vaccination in practice depends not only on the standard of the
vaccine but also on its being stored, transported and administered correctly.
(B47,
B210.89.w89,
D35.w1,
J3.102.w8,
J3.102.w9,
J13.24.w1,
J16.8.w1,
J16.22.w1,
J19.61.w1,
J35.148.w1, J64.21.w25,
J70.6.w1,
J70.8.w1,
J70.9.w1,
J70.10.w1,
J70.10.w2,
J70.12.w1,
J70.16.w2,
J70.17.w3,
W18.Apl01.sib1).
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| Data Source Notes |
DETAILED BACKGROUND INFORMATION "Vaccines prepared from virus grown in tissue culture
cells and suitably inactivated with an imine have proved effective when (a) their quality
is assured by adequate potency testing, (b) they are stored and transported at
refrigerator temperatures and (c) they are administered under the supervision of an
efficient veterinary service." (J70.6.w1).
Summary:
Production of inactivated vaccines requires: availability
of sufficient viral antigen, in high concentration; treatment to remove all infectivity;
addition of adjuvant to ensure effective stimulation of the animal's immune response,
without toxic effect (J64.21.w25,
J70.9.w1).
- Modern inactivated vaccines are grown in cell culture, inactivated using
acetylethylenimine or binary ethylenimine, and emulsified with adjuvant such as aluminium
hydroxide, or oil (B47,
B210.89.w89).
- Antigen may be concentrated such that dose volumes of e.g. 5ml for
trivalent vaccines may be produced (J70.9.w1).
- Inactivated antigen may be stored in a highly concentrated form at ultra-low
temperatures in vaccine banks (J70.8.w1,
J70.9.w1).
- Vaccines are available which have been shown to give a high level of immunity in cattle,
pigs and sheep within a few days of vaccination (D35.w1,
J70.12.w1,
J70.16.w2,
J70.17.w4).
Vaccine efficacy:
Vaccine efficacy depends on a number of factors including:
- The potency of vaccine itself.
- To be approved for use, FMD vaccines have to pass stringent potency tests to prove their
efficacy.
- May be tested by ability to protect cattle against the development of secondary lesions
on the feet following inoculation of FMDV into the tongue
(intradermolingual
challenge).
- Vaccine dose extinction point has been adopted by the European Pharmocopoeia (EP) as
their definitive reference method.
- May be tested by serological methods, e.g. mean virus neutralising titre.
- (J16.22.w1,
J70.10.w1,
J70.10.w2).
- Storage and transport.
- If vaccines are used after storage for too long or storage or transport in incorrect
conditions (e.g. allowed to get too warm) their efficacy decreases very significantly.
- optimum storage temp is 3 to 8 C. FMD vaccines must not be frozen
- This is most likely to be a problem in areas where rapid transport and cold storage
facilities are not reliably available.
- (J16.8.w1,
J70.6.w1,
B210.89.w89)
- Proper use:
- Efficacy is decreased if handling (e.g. gentle shaking of contents) and dosing (site,
quantity) are not carried out correctly.
- Administration under the supervision of an effective veterinary service provides the
best efficacy (J70.6.w1).
(Factors relating to the interaction between the vaccine and animals
(individuals and populations) are included on the Literature Reports: Vaccination Regimes page).
History of inactivated vaccine development:
- Initially, inactivated FMDV vaccines were developed by treating suspensions of virus
taken from naturally-infected animals with a dilute solution of formaldehyde
and/or by heating. Greater potency vaccines were developed by extracting virus from
infected tongue epithelium, adsorbing onto aluminium hydroxide gel followed by
controlled-temperature treatment with formaldehyde solution (J3.102.w8,
J16.8.w1).
Progress in virus growth and harvesting:
- Virus from naturally infected cattle:
- Lesion material or viraemic blood of naturally infected cattle was used as a source of
virus.
- Virus suspensions were produced by treatment of this natural material .
- Dose volumes of 30-100 mL were required.
- Vaccine potency of preparations from blood varied due to variations in
time of maximal viraemia in donor cattle
(J3.102.w8,
J16.8.w1).
- Virus from deliberately infected cattle:
- Virus was taken from tongue epithelium and vesicular fluid of
deliberately-infected cattle .
- Problems included:
- Requirement to infect cattle to produce virus.
- Limited production depending on handling of the infected cattle.
- Variation in time to maximal virus titre in different individuals.
- Risk of infected cattle to the surrounding livestock - isolation
facilities required.
- Requires disposal of infected carcasses
- Aesthetically undesirable to use live animals.
(J3.102.w8,
J16.8.w1, J64.21.w25)
- Virus from deliberately infected laboratory animals:
- Virus was grown in rabbits, hamsters, guinea-pigs, day-old chicks or
chicken eggs then inactivated.
- Variation in time to maximal virus titre in different individuals.
- Risk to nearby livestock - isolation facilities required
- Requires disposal of infected carcasses
- Aesthetically undesirable to use live animals.
(J3.102.w8,
J16.8.w1).
- Tissue culture:
- Frenkel culture:
- Epithelium and superficial tissues collected from the tongues of
freshly-slaughtered cattle.
- Virus grown in this tissue.
- Allows greater quantities of virus to be grown and processed; however,
increasing production to come with increased demand during an outbreak may be difficult
- Requires constant supply of fresh tongue epithelium; this may be obtained
relatively easily with good organisation of collection at slaughterhouses, but requires
co-operation of the slaughterhouse and good hygienic conditions.
- High and constant virus output may be achieved.
- No adaptation of virus to the culture system is required.
- Reduces risk of allergic reaction to foreign proteins.
- Simple, cheap medium for culture, not requiring any serum.
- Impossible to prevent low-level contamination with bacteria and yeast
(although product may be sterile following filtration).
(J3.102.w8,
J16.8.w1, J64.21.w25,
J70.9.w1).
- Growth of virus in primary tissue culture
- Calf kidney or pig kidney cells.
- Grown in monolayer.
- Requires constant supply of tissue for cells
- (J3.102.w8,
J16.8.w1, J70.9.w1).
- Growth of virus in continuous cell line culture
- BHK (baby hamster kidney) cells used.
- Growth in suspension possible, therefore greater production possible
- No requirement for fresh tissue
(J3.102.w8,
J16.8.w1, J64.21.w25,
J70.9.w1).
Progress in inactivation:
- Inactivation is one of the most important steps in producing a safe
efficacious FMD vaccine.
- Vaccine with any residual inactivity is unacceptable (J70.17.w3).
- Inactivation must not affect the integrity of the viral capsid. (J70.17.w3)
- With heat alone: inaccurate.
- With formaldehyde solution, 0.02-0.10%, buffered to pH 7.6-9.2,
heated to 23-26 °C for 24-48 hours.
- Inactivation does not follow first order kinetics.
- Problems have occurred with incompletely inactivated vaccines even
following prolonged treatment times
- e.g. treatment with 0.05% formalin, with incubation at 37 °C, may not
completely inactivate virus (J13.24.w1)
- Formaldehyde-inactivated vaccine preparations are stable and may
remain protective for 10 years or more.
- (J3.102.w8,
J13.24.w1,
J16.8.w1, J35.148.w1,
J64.21.w25,
J70.9.w1).
- Beta-propriolactone (BPL):
- Specially purified chemical required.
- Rapid, but care required to avoid sudden pH fall and complete
denaturation of viral antigen (J16.8.w1).
- Aziridines: acetylethyleneimine (AEI) and similar chemicals such as
binary ethylenimine (BEI) and propylenimine
- Acetylethyleneimine (AEI), 0.05% (J19.61.w1).
- Aziridine vaccine preparations have a relatively short shelf life
(e.g. up to two years).
- Stability can be improved by treatmwnt with formaldehyde. (J64.21.w25)
- Bromoethylamine hydrobromide (BEA), at pH
above 8 (at which it is
transformed to the active substance, ethylenimine) (J70.9.w1).
- More reliable.
- Less toxic than aziridines. (J64.21.w25)
- First-order kinetics
- "tailing off" of reaction may occur under certain conditions,
and testing to ensure inactivation is important (J70.9.w1).
- Acts directly on viral genome.
- Formaldehyde plus BEI, simultaneously.
- Synergistic effect, 100-fold increased rate of inactivation;
inactivation can be achieved within eight hours. (J64.21.w25)
(J3.102.w8,
J3.102.w9,
J16.8.w1,
J19.61.w1, J35.148.w1,
J70.9.w1, B210.89.w89)
Purification:
Purification of antigen is important. impurities present in a vaccine may cause both
local reactions at the site of vaccine inoculation, and systemic reactions.(J70.17.w3).
- Virus may be precipitated using polyethylene glycol (PEG) or polyethylene oxide
(PEO)
- Continuous centrifugation may be used for large scale collection of precipitated
antigen.(J70.9.w1).
- Filtration may also be used for collection of precipitated antigen.(J70.9.w1).
Adjuvants:
Adjuvants are essential to increase the immunogenicity of the antigen used in FMDV
vaccines (J70.17.w3).
Storage:
- Prepared vaccines need to be stored refrigerated, usually at 4 +/- 2
°C; at higher temperatures immunogenic activity is progressively lost.
(J70.17.w5)
- Freezing and thawing should be avoided, as the integrity of the
vaccines may be damaged and the oil emulsion or aluminium hydroxide gel
can be broken, again decreasing or destroying immunogenicity. (J70.17.w5)
- Concentrated inactivated FMDV antigens may be stored at ultralow temperatures which
allow stable storage for several years and formulation of vaccine when required (J70.16.w2).
- The European Union Vaccine Bank contains inactivated antigen of six different FMD virus
strains (O1 Manisa, O1 BFS, C Noville, Asia 1 Shamir, A22 Iraq, A24 Cruzeiro) sufficient
for the production of five million cattle doses of vaccine for each strain (W18.Apl01.sib1).
Quality control:
- Virus inactivation and follow-up safety tests are the most critical
steps in the preparation of inactivated FMD vaccines" (J64.21.w25)
- Vaccines must be quantified for their antigen content.
- Biological test systems may be used (originally cattle tongue and
suckling mice tests, later cell culture tests).
- Complement fixation tests, which may be used to give a quantitative
estimate of total antigenic mass or of just the major 140 S
immunogen.
- ELISA to estimate 140 S particles.
- Sucrose density gradient analysis. Quantitative test, estimating the 140
S antigen in µg/ml. Now accepted internationally as a standard test.
(J64.21.w25, J70.9.w1).
- Prior to vaccines being licensed for use, stringent tests must be passed
for safety and efficacy (J16.8.w1,
B210.89.w89).
- Testing using intradermolingual inoculation in cattle - prescribed by the
European Pharmacopoeia as a final proof of safety (J70.9.w1).
- "In vitro tests are more reliable than the intradermolingual test.
More antigen can be screened in one test and for detection of virus that was not released
from the cells, blind passages can be made" (J70.9.w1).
- In vitro testing of inactivated vaccines by infection of tissue
culture cells may be an effective alternative to testing using intradermolingual
inoculation of cattle, for all stages of testing before adjuvants are added (J19.68.w4)
- Inactivation kinetics may be used to indicate when total inactivation has
occurred, at least if the inactivation process follows first-order kinetics (J70.9.w1).
- Repeating inactivation following transfer of suspension to a second vessel may be used
to avoid the risk of particles avoiding contact with inactivant e.g. on container lids or
in tubes or valves (J64.21.w25,
J70.9.w1).
- Plaque assay in a sensitive cell culture system may be use for
titration of infectivity to verify the kinetics of virus
inactivation. (J64.21.w25)
- Potency testing (efficacy testing) is very important. It is also
expensive, requiring the use of live animals in biosecure facilities.
Testing to Ph. Eur standards involves injection of a full, 0.25 or 0.167
of a dose of vaccine into each of individually identified cattle,
followed by intradermolingual challenge three weeks later, then checking
eight days later to determine the percentage of cattle protected. The
number of cattle used in the test is minimised for reasons of cost and
welfare. Unfortunately, between-test variability is high, so that a
vaccine with theoretical PD50 value of 9.99 may have an actual value of
4.59 to 25.25. (J70.25.w6)
- For batch testing, serological testing for antibody levels at 21
days post vaccination is acceptable if a validated technique is used
for antibody level determination and a statistical correlation has
been established between antibody level and protection. (J70.25.w6)
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